Genetically Encoded Phage-Displayed Cyclic Peptides via Michael Addition
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Solution Overview
Problem
Existing methods for selecting and cyclizing phage-displayed peptides are limited by low viability in vivo due to disulfide bonds and non-selective organic linkers, leading to structural constraints and heterogeneity, which affects phage production and target binding efficacy.
Innovation Solution
A method involving the use of non-canonical amino acids, such as Nε-acryloyl-lysine, to form cyclic peptides through proximity-driven Michael addition reactions, allowing for higher phage viability and selective coupling in bacterial host cells, enabling the production and screening of cyclic peptides that bind specifically to desired targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If disulfide bonds or organic linkers are used to conjugate cysteines in phage-displayed peptides, then cyclic peptide structure is formed, but phage viability is limited and structural heterogeneity occurs
Solution Approach 1:
The patent changes the chemical parameters of cyclization by using non-canonical amino acids with electrophilic side chains (such as Nε-acryloyl-lysine) instead of traditional disulfide bonds or organic linkers. This enables cyclization through proximity-driven Michael addition reactions that occur spontaneously in bacterial host cells, forming cyclic peptides with improved structural homogeneity and phage viability.
Solution Approach 2:
The patent employs self-service by utilizing the bacterial host cell's translation machinery to incorporate non-canonical amino acids directly into the phage coat protein during synthesis. The cyclization occurs automatically through Michael addition reactions between the incorporated non-canonical amino acid and cysteine residues, eliminating the need for external chemical conjugation steps that compromise phage viability.
2Stability of the object's composition
If non-selective organic linkers are used for cysteine conjugation, then cyclic peptide formation is achieved, but structural constraints and heterogeneity affect phage production
Solution Approach 1:
The patent applies local quality by incorporating non-canonical amino acids with specific electrophilic properties at defined positions within the peptide sequence. This creates localized reactivity sites that selectively react with cysteine residues to form cyclic structures with consistent geometry, improving structural homogeneity and facilitating reliable phage production.
Solution Approach 2:
The patent substitutes mechanical/chemical conjugation systems (organic linkers requiring external reagents) with a biochemical system based on proximity-driven Michael addition reactions. This replacement eliminates the need for complex conjugation chemistry and enables straightforward phage production through standard bacterial transformation and expression protocols.
3Reliability
If traditional cyclization methods are used, then cyclic peptide binding is achieved, but in vivo use is limited due to low viability
Solution Approach 1:
The patent changes the chemical stability parameters of the cyclic peptide linkage by using covalent bonds formed through Michael addition reactions between non-canonical amino acids and cysteines. These bonds are more stable in vivo compared to disulfide bonds, enabling the cyclic peptides to maintain their structure and binding efficacy in living organisms.
Solution Approach 2:
The patent employs disposable bacterial host cells for the production and screening of phage particles displaying cyclic peptides. The system allows for rapid generation of phage libraries through bacterial transformation and expression, with the bacteria serving as disposable factories that can be easily discarded after phage production, enabling high-throughput screening for in vivo applications.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method achieves cyclic peptides with enhanced target affinity and stability, demonstrating up to six-fold stronger binding to protein targets compared to linear counterparts, with improved phage viability and reduced proteolysis.
Implementation Method 1
A method involving the use of non-canonical amino acids, such as Nε-acryloyl-lysine, to form cyclic peptides through proximity-driven Michael addition reactions
Data Source
AI summary
Embodiments of the present disclosure pertain to methods of selecting cyclic peptides that bind to a target by transforming a phage display library with a plurality of nucleic acids into bacterial host cells, where the nucleic acids include phage coat protein genes with a combinatorial region that encodes at least one cysteine and at least one non-canonical amino acid. The transformation results in the production of phage particles with phage coat proteins where the cysteine and the non-canonical amino acid couple to one another to form a cyclic peptide library. Phage particles are then screened against the desired target to select bound cyclic peptides. Amino acid sequences of the selected cyclic peptides are then identified. Additional embodiments pertain to methods of constructing a phage display library that encodes the cyclic peptides. Further embodiments of the present disclosure pertain to the produced cyclic peptides, phage display libraries and phage particles.